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| United States Patent |
5,149,829
|
|
Seib
, et al.
|
September 22, 1992
|
Method of preparing dry, concentrated salts of ascorbate 2-polyphosphate
Abstract
A method of preparing dried, concentrated ascorbate 2-polyphosphate salts
is provided wherein a starting aqueous solution containing the desired
2-polyphosphate species (as well as contaminating inorganic phosphates) as
sodium and potassium salts is subjected to a sequential precipitation
technique to first remove the contaminants, followed by recovery of the
desired species. In preferred practice, the inital starting solution is
first supplemented with calcium chloride and sufficient calcium hydroxide
to adjust the pH from about 6-8, thereby causing precipitation of the
inorganic contaminants. At this point the ascorbate 2-polyposphate species
are recovered from the supernatant and dried. The latter preferably
involves washing the precipitated organophosphate salts with an aqueous
organic solvent, concentration, addition of a non-solvent such as ethanol
to induce precipitation, and subsequent drying. The resulting product is a
concentrated salt form of ascorbate 2-polyphosphate that is a
free-flowing, dried powder which can be readily handled, stored and used
as an active vitamin C supplement in food or pharmacological systems.
| Inventors:
|
Seib; Paul A. (Manhattan, KS);
Wang; Xiao Y. (Manhattan, KS)
|
| Assignee:
|
Hoffmann-La Roche Inc. (Nutley, NJ)
|
| Appl. No.:
|
641042 |
| Filed:
|
January 14, 1991 |
| Current U.S. Class: |
549/222 |
| Intern'l Class: |
C07F 009/06 |
| Field of Search: |
549/222
|
References Cited
| Foreign Patent Documents |
| 0231496 | Sep., 1990 | JP.
| |
| 8700172 | Jan., 1987 | WO.
| |
Primary Examiner: Ivy; C. Warren
Assistant Examiner: Owens; Amelia A.
Attorney, Agent or Firm: Gould; George M., Epstein; William H.
Claims
We claim:
1. A method of preparing dried, concentrated ascorbate 2-polyphosphate
salts, comprising the steps of:
providing a quantity of ascorbate 2-polyphosphate species and inorganic
phosphate contaminants in aqueous solution;
adding a member selected from the group consisting of calcium chloride and
magnesium chloride to said solution, with the concentration of said
contaminants in said solution being sufficiently small to prevent
formation of a gel, with the pH of the solution being at a level of below
10;
precipitating at least a portion of said contaminants as the calcium or
magnesium salts thereof, with the ascorbate 2-polyphosphate species from
said reaction mixture remaining in the supernatant; and
recovering said ascorbate 2-polyphosphate species from said supernatant as
a dried product.
2. The method of claim 1, said contaminants being present in said solution
at a level of no more than about 1.5% by weight calculated as phosphorus.
3. The method of claim 2, said contaminants being present in said solution
at a level of no more than about 1.0% by weight.
4. The method of claim 3, said contaminants being present in said solution
at a level of no more than about 0.14% by weight.
5. The method of claim 1, wherein calcium chloride is added to said
solution, and base is added to the solution to adjust the pH thereof.
6. The method of claim 1, including the step of adjusting the temperature
of said solution after addition of said member to a level of from about
5.degree.-35.degree. C.
7. The method of claim 6, said level being from about 10.degree.-25.degree.
C.
8. The method of claim 1, the pH of said solution being at a level of from
about 6-8.
9. The method of claim 1, the molar ratio of ascorbate 2-polyphosphate
ester to calcium or magnesium ion in said solution being from about 1:2.5
to 1:4.
10. The method of claim 9, said ratio being about 1:3.5.
11. The method of claim 1, said recovering step comprising the steps of:
concentrating said supernatant;
adding to said concentrated supernatant a quantity of a non-solvent for
said ascorbate 2-polyphosphate species which is miscible in water, and
causing said species to precipitate; and
drying said precipitated ascorbate 2-polyphosphate species.
12. The method of claim 11, including the step of washing said precipitate
to remove chloride salts therefrom, prior to said drying step.
13. The method of claim 11, said non-solvent being selected from the group
consisting of methanol, ethanol, tetrahydrofuran, dioxane and acetone.
14. The method of claim 13, said non-solvent being ethanol, and being added
in a volume substantially equal to that of said concentrated supernatant.
15. The method of claim 12, said washing step comprising contacting said
precipitate with a mixture of water and ethanol.
16. The method of claim 15, said water-ethanol mixture being about a 1:1
v/v mixture.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is broadly concerned with a method of preparing dry,
concentrated salts of the stereoisomeric forms of ascorbate
2-polyphosphate (L-ascorbate and D-erythorbate), and particularly the
calcium and magnesium salts thereof, in good yields and with a high
proportion of contaminating inorganic species removed. More particularly,
it is concerned with such a method wherein aqueous solutions of sodium
and/or potassium L-ascorbate 2-polyphosphate are first treated by aqueous
dilution, addition of CaCl.sub.2 or MgCl.sub.2, and pH adjustment as
necessary in order to precipitate contaminating inorganic salts; the
supernatant is then concentrated, and a water-miscible organic solvent is
added to precipitate the desired organophosphate salts, and the final
precipitate is washed to remove chloride salts and dried.
2. Description of the Prior Art
U.S. Pat. No. 4,647,672 represents a significant breakthrough in the art
and describes stable, 2-polyphosphorylated species of L-ascorbic acid and
its stereoisomers. The 2-polyphosphate ester of L-ascorbate described in
this patent has proven to be an excellent source of vitamin C,
particularly in aquatic feeds. It is furthermore believed that compounds
of this type may have significant utility in human foods and
pharmacological preparations.
The subject patent describes preparation of ascorbate 2-polyphosphate in
liquid form, i.e., in aqueous solution. Such solutions contain significant
amounts of inorganic contaminants which render them less suitable in human
foods or other contexts requiring high vitamin potency or purity The '672
Patent discloses a conventional separation and purification technique for
obtaining a dry product, involving use of ion exchange column
chromatography. While this method does result in a dried, purified
product, it is unsuitable for large-scale commercial preparation of a
dried, powder-like product. The cost and technical difficulties associated
with ion exchange column chromatography of polyphosphorylated L-ascorbate
are formidable, and this method has not even been attempted on a
commercial scale, even though the advantages of a dried, concentrated
product are manifest.
There is accordingly a real and unsatisfied need in the art for an
improved, low cost method for the purification and separation of salts of
ascorbate 2-polyphosphate from aqueous solution which meets the twin goals
of providing a dried, concentrated, powder-like product by means adapted
for large-scale commercial production.
SUMMARY OF THE INVENTION
The present invention overcomes the problems described above and provides a
greatly improved method for preparing dried salts of L-ascorbate
2-polyphosphate; the method gives the desired end product in good yield,
with the elimination of a very high proportion of contaminating inorganic
salts. At the same time, the method is eminently suited for
commercial-scale operations and is low in cost.
Broadly speaking, the method of the invention involves initially providing
a quantity of sodium and/or potassium ascorbate 2-polyphosphate species in
aqueous solution, which also includes substantial amounts of inorganic
phosphate contaminants. Such a starting solution is prepared by the
methods described in U.S. Pat. No. 4,647,672, which is incorporated by
reference herein.
In the next step, a member selected from the group consisting of calcium
chloride and magnesium chloride is added to the starting solution, with
care being taken that the concentration of inorganic contaminants present
is sufficiently small to prevent formation of a gel. In this respect, if
the contaminant level is too great, the entire mixture sets into a
continuous gel rather than a discontinuous precipitate (inorganic salts)
suspended in a continuous liquid phase (solution of organophosphates). A
gel-like phase is very difficult to deal with further.
If calcium chloride is initially added, most of the inorganic phosphate
salts precipitate. The pH of the mixture should next be adjusted to a
level of about 6-8 by addition of calcium hydroxide, causing additional
inorganic phosphate salts to precipitate, while the desired ascorbate
2-polyphosphate species from the original reaction mixture remain
dissolved in the supernatant. On the other hand, if magnesium chloride is
initially added, no additional base is required to obtain the desired pH
level (about 7-8).
The final step of the method involves recovery of the ascorbate
2-polyphosphate species from the supernatant as a dried product. This may
be accomplished by a number of techniques, but most desirably the
supernatant is initially concentrated, where upon a non-solvent for the
ascorbate 2-polyphosphate species which is miscible in water (e.g.,
ethanol) is added to the concentrated supernatant. This causes
precipitation of the ascorbate 2-polyphosphate species. In order to remove
sodium and/or potassium chloride from the precipitated organophosphates,
the latter are contacted with a water-organic solvent mixture, and the
supernatant solution is removed and discarded. Upon drying, the
precipitated ascorbate 2-polyphosphate salt gives a free-flowing,
powder-like product which can be conventionally stored and used in a
variety of food systems for example.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In the first step of the invention, a quantity of sodium and/or potassium
ascorbate 2-polyphosphate species in aqueous solution is provided, and a
member selected from the group consisting of calcium chloride and
magnesium chloride is added thereto. Advantageously, the molar ratio of
ascorbate 2-polyphosphate to calcium or magnesium ion after such addition
should be from about 1:2.5 to 1:4, and more preferably about 1:3.5. In
order to inhibit the formation of an unworkable gel upon such addition,
the level of inorganic phosphate contaminants in the starting aqueous
solution should be no more than about 1.5% by weight as phosphorus, and
more preferably not more than about 1.0% by weight as phosphorus. In order
to achieve this level of contaminant concentration, it may be necessary to
dilute the aqueous phosphate solution with water. As a further step to
inhibit gel formation, the pH of the solution may be adjusted to a level
below about 10, and most preferably below about 8.
Upon addition of the calcium or magnesium chloride, steps are taken to
ensure the precipitation of at least a portion of the inorganic phosphate
contaminants present in the starting solution. When calcium chloride is
added, the pH is preferably adjusted as necessary to a level of about 6-8
by the addition of calcium hydroxide, but when magnesium chloride is
added, no base is required. In any event, the preferred pH of the solution
at this point is about 7. The mixture is then stirred for about 1-10
minutes, followed by a separation step, typically using centrifugation or
filtration techniques. The supernatant is then separated from the
precipitate by decantation, and the precipitated salts are serially washed
and centrifuged, and the resulting supernatant solutions are combined.
The conditions employed for removing the inorganic phosphate salts also
affect the proportions of the 2-triphosphate to 2-diphosphate to
2-monophosphate ester of L-ascorbate. To limit hydrolysis of the
2-polyphosphate esters to the 2-monophosphate ester during precipitation
and removal of calcium or magnesium inorganic phosphates, the pH of the
system should be maintained below about 10 and above about 6, and the
molar ratio of Ca.sup.+2 or Mg.sup.+2 to L-ascorbate should be as low as
possible to achieve precipitation of the inorganic phosphates. Moreover,
the temperature should be maintained at a level of from about
5.degree.-35.degree. C., most preferably from about 10.degree.-25.degree.
C. Furthermore, the initial reaction mixture should be diluted to a level
of about 0.5% by weight phosphorus concentration, and most preferably to
about 0.14% by weight phosphorus.
In order to recover the desired 2-polyphosphate species from the
supernatant, the latter is first concentrated, typically by vacuum,
whereupon a non-solvent for the desired ester species is added. This
non-solvent must be miscible with water, and is preferably taken from the
group consisting of methanol, ethanol, tetrahydrofuran, dioxane and
acetone. This serves to precipitate the desired phosphate ester species,
which can then be dried. Ethanol is the most preferred non-solvent, and is
normally added in a volume substantially equal to that of the concentrated
supernatant. Acetone is least preferred because of its volatility.
It is also desirable in most instances to remove sodium and/or potassium
chloride from the precipitated ester species, prior to the final drying
step. This can readily be accomplished by contacting the precipitate from
the concentrated supernatant with a 1:1 v/v mixture of water and ethanol.
The following example sets forth the most preferred method in accordance
with the invention. The example is provided for illustrative purposes
only, and nothing therein should be taken as a limitation upon the overall
scope of the invention.
EXAMPLE
A 400 ml beaker was fitted with a pH electrode, a magnetic stirring-bar,
and a buret. To the beaker, which was placed in a water bath at
33.degree.-35.degree. C., was added in sequence, water (105 ml),
L-ascorbic acid (30 g, 171 mmole, 1.63M) and 10M sodium and/or potassium
hydroxide to pH about 11.0. Sodium trimetaphosphate (95-97% pure, about
232 mmole) was added, and the pH maintained at 10.5-10.7 by periodic
addition of 10M sodium and/or potassium hydroxide. The reaction mixture
was stirred continuously and stopped after 24 hr. reaction time. The
reaction mixture, which had a total volume of about 200 ml, was diluted to
volume (500 ml.) with water.
A 25 ml aliquot of the above reaction mixture (8.5 meq. of ascorbic acid in
ester form) was mixed with 175 ml of water, and the mixture cooled to
10.degree. C. Ten mls of a concentrated aqueous solution containing 25
mmole of calcium chloride was added along with 5 ml of saturated aqueous
calcium hydroxide, with stirring for 6 minutes, followed by
centrifugation. The supernatant was then decanted, and the precipitated
inorganic salts were washed with water (3 times, 50 ml each) followed each
time by centrifugation. The four supernatant solutions (about 350 ml) were
then combined and vacuum evaporated below 50.degree. C. to about 40 ml,
and the concentrate was centrifuged to remove small amounts of inorganic
salts. The supernatant was diluted to 90 ml, where the mixture includes
about 0.1M ascorbic acid equivalents.
An equal volume of ethanol was added to the concentrated solution to
precipitate the organophosphate salts. These precipitates were collected
by four successive collection steps of centrifugation and washing with a
1:1 water/ethanol solution. The precipitated organic salts were then dried
under reduced pressure (e.g., 50 torr) over phosphorus pentoxide.
The solid salts of L-ascorbate 2-polyphosphate contained a 3:1 molar ratio
of Ca.sup.+2 :Na.sup.+1 as determined by elemental assay, and the salt
accounted for 87% by weight of the ascorbic acid equivalents in the
original aqueous reaction mixture, as determined by UV at 258 nm and pH 10
using absorbtivity 16.times.10.sup.3 mol/g cm. UV and high performance
liquid chromatography showed the 3:1 Ca.sup.+2 :Na.sup.+1 salt contained
78% by weight of a 1:1.86:4.50 molar mixture of L-ascorbate
2-monophosphate:L-ascorbate 2-diphosphate:L-ascorbate 2-triphosphate. The
product was also found to contain 5% by weight moisture, 3% ethanol, 2%
calcium orthophosphate, and less than 1% of calcium pyro-, tripoly-, or
trimetaphosphate by ion-exchange chromatography. The product showed a
negative chloride test when an aqueous solution thereof was treated with
silver ions.
The following is a flow chart outlining the above-described procedure,
which also includes a mass balance. The abbreviations used in the flow
chart are also indentified below.
##STR1##
Ca.sub.1.1 Na.sub.0.8 AsMP/Ca.sub.1.5 Na.sub.1.0 AsDP/Ca.sub.1.9
Na.sub.1.3 AsTP=1:1.9:4.5 (mole ratio)
Recovery of reacted AsA determined by UV=87%
Purity of determined by UV=78%
Expected dry mass of inorganic and organic phosphate salts=5.18 g
Found dry mass of inorganic and organic phosphate salts=5.10 g
______________________________________
AsA L-ascorbic acid
AsMP L-ascorbate 2-monophosphate
AsDP L-ascorbate 2-diphosphate
AsTP L-ascorbate 2-triphosphate
STMP Sodium trimetaphosphate
4-5-ene tetraP
4,5-Dehydrated L-ascorbate 2-triphosphate
6-phosphate
Pi Orthophosphate, sodium salt
PPi Pyrophosphate, sodium salt
PPPi Tripolyphosphate, sodium salt
TrimetaPPPi
Trimetaphosphate, sodium salt
CaPi Orthophosphate, calcium salt
CaPPi Pyrophosphate, calcium salt
CaPPPi Tripolyphosphate, calcium salt
Ca/Na AsPP
Calcium sodium L-ascorbate 2-polyphosphate
______________________________________
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